Insulin analogues with chlorinated amino acids and nucleic acids encoding the same

ABSTRACT

An insulin analog comprises a B-chain polypeptide incorporating a chlorinated phenylalanine. The chlorinated phenylalanine may be located at position B24. The chlorinated phenylalanine may be para-monochloro-phenylalanine. The analog may be of a mammalian insulin, such as human insulin. A nucleic acid encodes such an insulin analog. The chlorinated insulin analogs retain significant activity. A method of treating a patient comprises administering a physiologically effective amount of the insulin analog or a physiologically acceptable salt thereof to a patient. Chlorine substitution-based stabilization of insulin may reduce fibrillation and thereby enhance the treatment of diabetes mellitus in regions of the developing world lacking refrigeration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of co-pending U.S.application Ser. No. 13/515,192 which was filed Jul. 18, 2012, which isa national stage application of PCT/US2010/060085 which was filed onDec. 13, 2010, which is a non-provisional of U.S. Patent Application No.61/285,955 which was filed on Dec. 11, 2009.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support awarded by the NationalInstitutes of Health under grant numbers DK040949 and DK074176. The U.S.government has certain rights to the invention.

TECHNICAL FIELD

This invention relates to polypeptides that are resistant to thermaldegradation. More particularly, this invention relates to thermallystabilized insulin analogues. Even more particularly, this inventionrelates to insulin analogues that are chemically and thermallystabilized by the incorporation of the element chlorine into an aminoacid of the insulin analogue. Chlorine is classified as a halogen and isdistinguished from the ordinary constituents of proteins by its atomicradius, electronegativity, stereoelectronic distribution of partialcharges, and transmitted effects on the stereoelectronic properties ofneighboring atoms in a molecule.

BACKGROUND OF THE INVENTION

The engineering of ultra-stable proteins, including therapeutic agentsand vaccines, may have broad societal benefits in regions of thedeveloping world where electricity and refrigeration are notconsistently available. An example of a therapeutic protein susceptibleto thermal degradation is provided by insulin. The challenge posed byits chemical and physical degradation is deepened by the pendingepidemic of diabetes mellitus in Africa and Asia. Because chemicaldegradation rates of insulin analogues correlate inversely with theirrelative stabilities, the design of ultra-stable formulations mayenhance the safety and efficacy of insulin replacement therapy in suchchallenged regions.

The utility of some halogen substitutions in small organic molecules isknown in medicinal chemistry. Fluorinated functional groups are criticalto the efficacy of such widely prescribed small molecules asatorvastatin (Liptor™), an inhibitor of cholesterol biosynthesis, andfluoxetine hydrochloride (Prozac™), a selective serotonin reuptakeinhibitor used in the treatment of depression and other affectivedisorders. Although the atomic radius of fluorine is similar to that ofhydrogen, its large inductive effects modify the stereo-electronicproperties of these drugs, in turn enhancing their biologicalactivities. Similar considerations of physical organic chemistry pertainto the incorporation of larger halogen atoms, such as chlorine. Thesmall molecule montelukast sodium (Singulair™) is a leukotrieneinhibitor whose pharmaceutical properties are enhanced by covalentincorporation of a chlorine atom. Additionally, the use offluorine-substituted amino acids in an insulin analogue is provided inInternational Patent Application No. PCT/US2009/52477 filed 31 Jul.2009.

Modulation of the chemical, physical, and biological properties ofproteins by the site-specific incorporation of chlorine atoms intomodified amino acids are less well characterized in the scientificliterature than are the above effects of incorporation of fluorineatoms.

Aromatic side chains may engage in a variety of weakly polarinteractions, involving not only neighboring aromatic rings but alsoother sources of positive- or negative electrostatic potential. Examplesinclude main-chain carbonyl- and amide groups in peptide bonds.

Administration of insulin has long been established as a treatment fordiabetes mellitus. Insulin is a small globular protein that plays acentral role in metabolism in vertebrates. Insulin contains two chains,an A chain, containing 21 residues and a B chain containing 30 residues.The hormone is stored in the pancreatic β-cell as a Zn²⁺-stabilizedhexamer, but functions as a Zn²⁺-free monomer in the bloodstream.Insulin is the product of a single-chain precursor, proinsulin, in whicha connecting region (35 residues) links the C-terminal residue of Bchain (residue B30) to the N-terminal residue of the A chain (FIG. 1A).Although the structure of proinsulin has not been determined, a varietyof evidence indicates that it consists of an insulin-like core anddisordered connecting peptide (FIG. 1B). Formation of three specificdisulfide bridges (A6-11, A7-B7, and A20-B19; FIGS. 1A and 1B) isthought to be coupled to oxidative folding of proinsulin in the roughendoplasmic reticulum (ER). Proinsulin assembles to form solubleZn²⁺-coordinated hexamers shortly after export from ER to the Golgiapparatus. Endoproteolytic digestion and conversion to insulin occurs inimmature secretory granules followed by morphological condensation.Crystalline arrays of zinc insulin hexamers within mature storagegranules have been visualized by electron microscopy (EM).

Extensive X-ray crystallographic studies have been undertaken ofZn²⁺-coordinated insulin hexamers, the physiological storage form.Multiple crystal forms have been described in vitro, defining threestructural families, designated T₆, T₃R^(f) ₃ and R₆. In these hexamerstwo Zn ions are believed to lie along the central axis of the hexamer,each coordinated by three histidines (HisB10); additional low-affinityZn-binding sites have been observed in some crystal forms. The T-stateprotomer resembles the structure of an insulin monomer in solution. TheR-state protomer exhibits a change in the secondary structure of theB-chain: the central α-helix extends to B1 (the R state) or to B3(frayed R^(f) state).

Insulin functions in the bloodstream as a monomer, and yet it is themonomer that is believed to be most susceptible to fibrillation and mostforms of chemical degradation. The structure of an insulin monomer,characterized in solution by NMR, is shown in FIG. 1D. The A-chainconsists of an N-terminal α-helix (residues A1-A8), non-canonical turn(A9-A12), second α-helix (A12-A18), and C-terminal extension (A19-A21).The B chain contains an N-terminal arm (B1-B6), β-turn (B7-B10), centralα-helix (B9-B19), β-turn (B20-B23), β-strand (B24-B28), and flexibleC-terminal residues B29-B30. The two chains pack to form a compactglobular domain stabilized by three disulfide bridges (cystines A6-A11,A7-B7, and A20-B19).

Absorption of regular insulin is limited by the kinetic lifetime of theZn-insulin hexamer, whose disassembly to smaller dimers and monomers isrequired to enable transit through the endothelial lining ofcapillaries. The essential idea underlying the design of Humalog® andNovolog® is to accelerate disassembly. This is accomplished bydestabilization of the classical dimer-forming surface (the C-terminalanti-parallel β-sheet). Humalog® contains substitutions ProB28→Lys andLysB29→Pro, an inversion that mimics the sequence of IGF-I. Novolog®contains the substitution ProB28→Asp. Although the substitutions impairdimerization, the analogs are competent for assembly of a phenol- ormeta-cresol-stabilized zinc hexamer. This assembly protects the analogfrom fibrillation in the vial, but following subcutaneous injection, thehexamer rapidly dissociates as the phenol (or m-cresol) and zinc ionsdiffuse away. The instability of these analogs underlies their reducedshelf life on dilution by the patient or health-care provider. It wouldbe useful for an insulin analogue to augment the intrinsic stability ofthe insulin monomer while retaining the variant dimer-related β-sheet ofHumalog®.

Use of zinc insulin hexamers during storage is known and represents aclassical strategy to retard physical degradation and chemicaldegradation of a formulation in the vial or in the reservoir of a pump.Because the zinc insulin hexamer is too large for immediate passage intocapillaries, the rate of absorption of insulin after subcutaneousinjection is limited by the time required for dissociation of hexamersinto smaller dimers and monomer units. Therefore, it would advantageousfor an insulin analogue to be both (a) competent to permit hexamerassembly at high protein concentration (as in a vial or pump) and yet(b) sufficiently destabilized at the dimer interface to exhibitaccelerated disassembly—hence predicting ultra-rapid absorption from thesubcutaneous depot. These structural goals walk a fine line betweenstability (during storage) and instability (following injection).

Amino-acid substitutions in insulin have been investigated for effectson thermodynamic stability and biological activity. No consistentrelationship has been observed between stability and activity. Whereassome substitutions that enhance thermodynamic stability also enhancebinding to the insulin receptor, other substitutions that enhancestability impede such binding. The effects of substitution of Thr^(A8)by several other amino acids has been investigated in wild-type humaninsulin and in the context of an engineered insulin monomer containingthree unrelated substitutions in the B-chain (His^(B10)→Asp,Pro^(B28)→Lys, and Lys^(B29)→Pro) have been reported. Examples are alsoknown in the art of substitutions that accelerate or delay the timecourse of absorption. Such substitutions (such as Asp^(B28) in Novalog®and [Lys^(B28), Pro^(B29)] in Humalog®) can be and often are associatedwith more rapid fibrillation and poorer physical stability. Indeed, inone study a series of ten analogues of human insulin was tested forsusceptibility to fibrillation, including Asp^(B28)-insulin andAsp^(B10)-insulin. All ten were found to be more susceptible tofibrillation at pH 7.4 and 37° C. than is human insulin. The tensubstitutions were located at diverse sites in the insulin molecule andare likely to be associated with a wide variation of changes inclassical thermodynamic stability. Although a range of effects has beenobserved, no correlation exists between activity and thermodynamicstability.

Insulin is a small globular protein that is highly amenable to chemicalsynthesis and semi-synthesis, which facilitates the incorporation ofnonstandard side chains. Insulin contains three phenylalanine residues(positions B1, B24, and B25) and a structurally similar tyrosine atposition B26. Conserved among vertebrate insulins and insulin-likegrowth factors, the aromatic ring of Phe^(B24) packs against (but notwithin) the hydrophobic core to stabilize the super-secondary structureof the B-chain. Phe^(B24) lies at the classical receptor-binding surfaceand has been proposed to direct a change in conformation on receptorbinding. Phe^(B25) projects from the surface of the insulin monomerwhereas Tyr^(B26) packs near aliphatic side chains (Ile^(A2), Val^(A3),and Val^(B12)) at one edge of the core. The B24-related conformationalchange is proposed to enable Phe^(B25) and Tyr^(B26) to contact distinctdomains of the insulin receptor.

The present theory of protein fibrillation posits that the mechanism offibrillation proceeds via a partially folded intermediate state, whichin turn aggregates to form an amyloidogenic nucleus. In this theory, itis possible that amino-acid substitutions that stabilize the nativestate may or may not stabilize the partially folded intermediate stateand may or may not increase (or decrease) the free-energy barrierbetween the native state and the intermediate state. Therefore, thecurrent theory indicates that the tendency of a given amino-acidsubstitution in the insulin molecule to increase or decrease the risk offibrillation is highly unpredictable.

Fibrillation, which is a serious concern in the manufacture, storage anduse of insulin and insulin analogues for diabetes treatment, is enhancedwith higher temperature, lower pH, agitation, or the presence of urea,guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drugregulations demand that insulin be discarded if fibrillation occurs at alevel of one percent or more. Because fibrillation is enhanced at highertemperatures, diabetic individuals optimally must keep insulinrefrigerated prior to use. Fibrillation of insulin or an insulinanalogue can be a particular concern for diabetic patients utilizing anexternal insulin pump, in which small amounts of insulin or insulinanalogue are injected into the patient's body at regular intervals. Insuch a usage, the insulin or insulin analogue is not kept refrigeratedwithin the pump apparatus and fibrillation of insulin can result inblockage of the catheter used to inject insulin or insulin analogue intothe body, potentially resulting in unpredictable blood glucose levelfluctuations or even dangerous hyperglycemia. At least one recent reporthas indicated that lispro insulin (an analogue in which residues B28 andB29 are interchanged relative to their positions in wild-type humaninsulin; trade name Humalog®) may be particularly susceptible tofibrillation and resulting obstruction of insulin pump catheters.

Insulin fibrillation is an even greater concern in implantable insulinpumps, where the insulin would be contained within the implant for 1-3months at high concentration and at physiological temperature (i.e., 37°C.), rather than at ambient temperature as with an external pump.Additionally, the agitation caused by normal movement would also tend toaccelerate fibrillation of insulin. In spite of the increased potentialfor insulin fibrillation, implantable insulin pumps are still thesubject of research efforts, due to the potential advantages of suchsystems. These advantages include intraperitoneal delivery of insulin tothe portal circulatory system, which mimics normal physiologicaldelivery of insulin more closely than subcutaneous injection, whichprovides insulin to the patient via the systemic circulatory system.Intraperitoneal delivery provides more rapid and consistent absorptionof insulin compared to subcutaneous injection, which can providevariable absorption and degradation from one injection site to another.Administration of insulin via an implantable pump also potentiallyprovides increased patient convenience. Whereas efforts to preventfibrillation, such as by addition of a surfactant to the reservoir, haveprovided some improvement, these improvements have heretofore beenconsidered insufficient to allow reliable usage of an implanted insulinpump in diabetic patients outside of strictly monitored clinical trials.

As noted above, the developing world faces a challenge regarding thesafe storage, delivery, and use of drugs and vaccines. This challengecomplicates the use of temperature-sensitive insulin formulations inregions of Africa and Asia lacking consistent access to electricity andrefrigeration, a challenge likely to be deepened by the pending epidemicof diabetes in the developing world. Insulin exhibits an increase indegradation rate of 10-fold or more for each 10° C. increment intemperature above 25° C., and guidelines call for storage attemperatures <30° C. and preferably with refrigeration. At highertemperatures insulin undergoes both chemical degradation (changes incovalent structure such as formation of iso-aspartic acid, rearrangementof disulfide bridges, and formation of covalent polymers) and physicaldegradation (non-native aggregation and fibrillation).

Amino-acid substitutions have been described in insulin that stabilizethe protein but augment its binding to the insulin receptor (IR) and itscross-binding to the homologous receptor for insulin-like growth factors(IGFR) in such a way as to confer a risk of carcinogenesis. An exampleknown in the art is provided by the substitution of His^(B10) byaspartic acid. Although Asp^(B10)-insulin exhibits favorablepharmaceutical properties with respect to stability andpharmacokinetics, its enhanced receptor-binding properties wereassociated with tumorigenesis in Sprague-Dawley rats. Although there aremany potential substitutions in the A- or B chains that can beintroduced into Asp^(B10)-insulin or related analogues to reduce itsbinding to IR and IGFR to levels similar to that of human insulin, suchsubstitutions generally impair the stability of insulin (or insulinanalogues) and increase its susceptibility to chemical and physicaldegradation. It would be desirable to discover a method of modificationof insulin and of insulin analogues that enabled “tuning” ofreceptor-binding affinities while at the same time enhancing stabilityand resistance to fibrillation. Such applications would require a set ofstabilizing modifications that reduce binding to IR and IGFR to varyingextent so as to offset the potential carcinogenicity of analogues thatare super-active in their receptor-binding properties.

Therefore, there is a need for alternative insulin analogues, includingthose that are stable during storage but are simultaneously fast-acting.

SUMMARY OF THE INVENTION

It is, therefore, an aspect of the present invention to provide aninsulin analogue that provides altered properties, such as improvedstability, by chlorine substitution in an amino acid, where the analoguealso maintains at least a portion of biological activity of thecorresponding non-chlorinated insulin or insulin analogue.

In addition or in the alternative, it is an aspect of the presentinvention to provide an insulin analogue that is a fast acting insulinbut also has improved stability over previous fast-acting insulinanalogues.

In general, the present invention provides an insulin analoguecomprising a B-chain polypeptide which incorporates a chlorinated aminoacid. In one embodiment, the chlorinated amino acid is phenylalanine atposition B24. In one particular embodiment, the chlorinatedphenylalanine is para-monochloro-phenylalanine. In addition or in thealternative, the insulin analogue may be a mammalian insulin analogue,such as an analogue of human insulin. In one particular set ofembodiments, the B-chain polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 4-8 and polypeptideshaving three or fewer additional amino acid substitutions thereof.

Also provided is a nucleic acid encoding an insulin analogue comprisinga B-chain polypeptide that incorporates a chlorinated phenylalanine atposition B24 such as para-monochloro-phenylalanine. In one example, thechlorinated phenylalanine is encoded by a stop codon, such as thenucleic acid sequence TAG. An expression vector may comprise such anucleic acid and a host cell may contain such an expression vector.

The invention also provides a method of treating a patient. The methodcomprises administering a physiologically effective amount of an insulinanalogue or a physiologically acceptable salt thereof to the patient,wherein the insulin analogue or a physiologically acceptable saltthereof contains a chlorinated amino acid. In one embodiment, an insulinB-chain incorporates a chlorinated phenylalanine at position B24. In oneparticular embodiment, the chlorinated phenylalanine ispara-monochloro-phenylalanine. In addition or in the alternative, theinsulin analogue may a mammalian insulin analogue, such as an analogueof human insulin. Furthermore, the B-chain polypeptide may comprise anamino acid sequence selected from the group consisting of SEQ ID NOS:4-8 and polypeptides having three or fewer additional amino-acidsubstitutions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the sequence of humanproinsulin including the A- and B-chains and the connecting region shownwith flanking dibasic cleavage sites (filled circles) and C-peptide(open circles).

FIG. 1B is a structural model of proinsulin, consisting of aninsulin-like moiety and a disordered connecting peptide (dashed line).

FIG. 1C is a schematic representation of the sequence of human insulinindicating the position of residue B24 in the B-chain.

FIG. 1D is a ribbon model of an insulin monomer showing aromatic residueof Phe^(B24) in relation to the three disulfide bridges. The adjoiningside chains of Leu^(B15) (in black) and Phe^(B24) (in gray) are shown.The A- and B-chain chains are shown, and the disulfide bridges as ballsand sticks.

FIG. 1E is a space-filling model of insulin showing the Phe^(B24) sidechain within a pocket at the edge of the hydrophobic core.

FIG. 2A is a representation of ortho-monochlorinated-phenylalanine(2Cl-Phe).

FIG. 2B is a representation of meta-monochlorinated-phenylalanine(3Cl-Phe).

FIG. 2C is a representation of para-monochlorinated-phenylalanine(4Cl-Phe).

FIG. 3A is a graph showing the results of in vitro receptor-bindingassays using isolated insulin receptor (isoform B): human insulin(triangles), KP-insulin (squares), and 4Cl-Phe^(B24)-KP-insulin(inverted triangles).

FIG. 3B is a graph showing the results of in vitro receptor-bindingassays employing IGF-1R: human insulin (triangles), KP-insulin(squares), 4Cl-Phe^(B24)-KP-insulin (inverted triangles), and nativeIGF-I (circles).

FIG. 3C is a graph comparing the results of in vitro receptor-bindingassays using isolated insulin receptor (isoform B): human insulin (solidline), KP-insulin (dashed line), 4Cl-Phe^(B24)-KP-insulin (triangles)4F-Phe^(B24)-KP-insulin (squares).

FIG. 3D is a graph comparing the results of in vitro receptor-bindingassays using isolated insulin receptor (isoform B): human insulin (solidline), KP-insulin (dashed line), 4Cl-Phe^(B26)-KP-insulin (triangles)4F-Phe^(B26)-KP-insulin (squares).

FIG. 4 is a graph showing the hypoglycemic action of subcutaneous of4Cl-Phe^(B24)-KP-insulin in STZ induced diabetic Lewis rats over time(inverted triangles) relative to diluent alone (circles), human insulin(crosses), and KP-insulin (squares).

FIGS. 5A-C are graphs showing averaged traces of insulin cobaltsolutions showing characteristic spectral profiles from 400-750 nmbefore and after addition of 2 mM EDTA. Samples were dissolved in 50 mMTris (pH 7.4), 50 mM phenol, and 0.2 mM CoCl₂. NaSCN was then added to afinal concentration of 1 mM. Solid lines show data pre-EDTA extraction.Dashed lines show data post-EDTA extraction. Panel A: wild type insulin;Panel B: KP-insulin; Panel C; 4Cl-Phe^(B24)-KP-insulin.

FIG. 5D is a graph showing the kinetics of hexamer dissociation afteraddition of 2 mM EDTA as monitored at 574 nm (25° C. and pH 7.4). Datawere normalized to time zero for each sample: wild type (solid line),KP-insulin (dashed line), and 4Cl-Phe^(B24)-KP-insulin (dotted line).

FIG. 6 is a graph showing a plot of the mean filtered glucose infusionrate versus time after insulin dose for KP-insulin (Lispro insulin) and4Cl-Phe^(B24)-KP-insulin (4-Cl-Lispro insulin) at a dosage of 0.2 Unitsper kilogram of bodyweight.

FIG. 7 is a bar graph summarizing 20 pharmacodynamic studies in pigsdemonstrating significant improvement in ½ T-max late in4Cl-Phe^(B24)-KP-insulin over KP-insulin at five different dosinglevels.

FIG. 8 is a bar graph summarizing 14 pharmacodynamic studies in pigssuggesting improvement in ½ T-max early in 4Cl-Phe^(B24)-KP-insulin overKP-insulin at three different dosing levels.

FIG. 9 is a summary of ten, matched pharmacodynamics studies comparingthe relative potencies 4Cl-Phe^(B24)-KP-insulin with that of KP insulinas measured by area under the curve (AUC) in which the slightly reducedaverage potency for 4-Cl-KP was found not to be statisticallysignificant (p=0.22).

DESCRIPTION OF EMBODIMENTS

The present invention is directed an insulin analogue that providesgreater stability by chlorine substitution in an amino acid, where theanalogue then maintains at least a portion of biological activity of thecorresponding non-chlorinated insulin or insulin analogue. Particularly,the present invention provides insulin analogues that provide greaterstability by substitution of chlorine in an amino acid, where theanalogue then maintains at least a portion of biological activity of thecorresponding non-chlorinated insulin or insulin analogue. In oneparticular embodiment, the present invention provides insulin analoguesthat contain a para-monochloro-phenylalanine (4Cl-Phe^(B24)) residue asa substitution for wild type phenylalanine at position B24.

The present invention is not limited, however, to human insulin and itsanalogues. It is also envisioned that these substitutions may also bemade in animal insulins such as porcine, bovine, equine, and canineinsulins, by way of non-limiting examples.

Furthermore, in view of the similarity between human and animalinsulins, and use in the past of animal insulins in human diabeticpatients, it is also envisioned that other minor modifications in thesequence of insulin may be introduced, especially those substitutionsconsidered “conservative.” For example, additional substitutions ofamino acids may be made within groups of amino acids with similar sidechains, without departing from the present invention. These include theneutral hydrophobic amino acids: Alanine (Ala or A), Valine (Val or V),Leucine (Leu or L), Isoleucine (Ile or I), Proline (Pro or P),Tryptophan (Trp or W), Phenylalanine (Phe or F) and Methionine (Met orM). Likewise, the neutral polar amino acids may be substituted for eachother within their group of Glycine (Gly or G), Serine (Ser or S),Threonine (Thr or T), Tyrosine (Tyr or Y), Cysteine (Cys or C),Glutamine (Glu or Q), and Asparagine (Asn or N). Basic amino acids areconsidered to include Lysine (Lys or K), Arginine (Arg or R) andHistidine (His or H). Acidic amino acids are Aspartic acid (Asp or D)and Glutamic acid (Glu or E). Unless noted otherwise or wherever obviousfrom the context, the amino acids noted herein should be considered tobe L-amino acids. In one example, the insulin analogue of the presentinvention contains three or fewer conservative substitutions other thanthe 4Cl-Phe^(B24) substitution of the present invention. In anotherexample, the insulin analogue of the present invention contains one orfewer conservative substitutions other than the 4Cl-Phe^(B24)substitution of the present invention.

As used in this specification and the claims, various amino acids ininsulin or an insulin analogue may be noted by the amino acid residue inquestion, followed by the position of the amino acid, optionally insuperscript. The position of the amino acid in question includes the Aor B chain of insulin where the substitution is located. Thus, Phe^(B24)denotes a phenylalanine at the twenty fourth amino acid of the B chainof insulin, while Phe^(B25) denotes a phenylalanine at the twenty fifthamino acid of the B chain of insulin. A chlorinated amino acid may beindicated with the prefix “Cl—.” Therefore, chlorinated phenylalaninemay be abbreviated “Cl-Phe.” In the case of phenylalanine, the positionof the chlorine substituents or substituents on the phenyl side chainmay be further indicated by the number of the carbon to which thechlorine is attached. Therefore, ortho-monochloro-phenylalanine (shownin FIG. 2B) is abbreviated “2Cl-Phe,” meta-monochloro-phenylalanine(shown in FIG. 2C) is abbreviated “3Cl-Phe” andpara-monochloro-phenylalanine (shown in FIG. 2D) is abbreviated 4Cl-Phe.

The phenylalanine at B24 is an invariant amino acid in functionalinsulin and contains an aromatic side chain. The biological importanceof Phe^(B24) in insulin is indicated by a clinical mutation (Ser^(B24))causing human diabetes mellitus. As illustrated in FIGS. 1D and 1E, andwhile not wishing to be bound by theory, Phe^(B24) is believed to packat the edge of a hydrophobic core at the classical receptor bindingsurface. The models are based on a crystallographic protomer (2-Znmolecule 1; Protein Databank identifier 4INS). Lying within theC-terminal β-strand of the B-chain (residues B24-B28), Phe^(B24) adjoinsthe central α-helix (residues B9-B19). One face and edge of the aromaticring sit within a shallow pocket defined by Leu^(B15) and Cys^(B19); theother face and edge are exposed to solvent (FIG. 1E). This pocket is inpart surrounded by main-chain carbonyl and amide groups and so creates acomplex and asymmetric electrostatic environment.

It is envisioned that the substitutions of the present invention may bemade in any of a number of existing insulin analogues. For example, thechlorinated Phe^(B24) substitution provided herein may be made ininsulin analogues such as Lispro (KP) insulin, insulin Aspart, othermodified insulins or insulin analogues, or within various pharmaceuticalformulations, such as regular insulin, NPH insulin, lente insulin orultralente insulin, in addition to human insulin. Aspart insulincontains an Asp^(B28) substitution and is sold as Novalog® whereasLispro insulin contains Lys^(B28) and Pro^(B29) substitutions and isknown as and sold under the name Humalog®. These analogues are describedin U.S. Pat. Nos. 5,149,777 and 5,474,978. Both of these analogues areknown as fast-acting insulins.

While not wishing to be bound by theory, the chloro substitution at thepara position of an aromatic ring is believed to be buried within thedimer interface (4Cl-Phe^(B24)) and is also believed to acceleratehexamer disassembly by creating an unfavorable alignment ofchloro-aromatic electrostatic dipole moments, pairwise juxtaposed ateach dimer interface within the hexamer. This is believed to permit theinsulin analogue to be formulated in the presence of Zn²⁺ ions and stillretain the ability to be a fast-acting (meal time) insulin analogue.

A chlorinated-Phe substitution, including one at B24, may also beintroduced into analogues of human insulin that, while not previouslyclinically used, are still useful experimentally, such as DKP insulin,described more fully below, or miniproinsulin, a proinsulin analoguecontaining a dipeptide (Ala-Lys) linker between the A chain and B chainportions of insulin in place of the normal 35 amino acid connectingregion between the C-terminal residue of the B chain and the N-terminalresidue of the A chain (See FIG. 1B). Incorporation of chlorinated-Pheat position B24 in DKP-insulin (or other insulin analogues that containAsp^(B10) or that exhibit receptor-binding affinities higher than thatof human insulin) can reduce their receptor-binding affinities to besimilar to or below that of human insulin and so potentially enableclinical use. In this manner the cross-binding of insulin analogues tothe mitogenic IGFR may also be reduced.

The amino-acid sequence of human proinsulin is provided, for comparativepurposes, as SEQ ID NO: 1.

(proinsulin) SEQ ID NO: 1Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr- Cys-Asn

The amino acid sequence of the A chain of human insulin is provided asSEQ ID NO: 2.

(A chain) SEQ ID NO: 2 Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn

The amino acid sequence of the B chain of human insulin is provided asSEQ ID NO: 3.

(B chain) SEQ ID NO: 3 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Pro-Lys-Thr

Further combinations of other substitutions are also within the scope ofthe present invention. It is also envisioned that the substitutions ofthe present invention may also be combined with substitutions of priorknown insulin analogues. For example, the amino acid sequence of ananalogue of the B chain of human insulin containing the Lys^(B28)Pro^(B29) substitutions of lispro insulin (Humalog®), in which thechlorinated Phe substitution may also be introduced, is provided as SEQID NO: 4. Likewise, the amino acid sequence of an analogue of the Bchain of human insulin containing the Asp^(B28) substitution of aspartinsulin, in which a chlorinated-Phe^(B24) substitution may also beintroduced, is provided as SEQ ID NO: 5.

SEQ ID NO: 4 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Lys-Pro-ThrSEQ ID NO: 5 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Asp-Lys-Thr

A chlorinated-Phe^(B24) substitution may also be introduced incombination with other insulin analogue substitutions such as analoguesof human insulin containing His substitutions at residues A4, A8 and/orB1 as described more fully in co-pending International Application No.PCT/US07/00320 and U.S. application Ser. No. 12/160,187, the disclosuresof which are incorporated by reference herein. For example, a4Cl-Phe^(B24) substitution may be present with [His^(A4), His^(A8)],and/or His^(B1) substitutions in an insulin analogue or proinsulinanalogue having the amino acid sequence represented by SEQ ID NO: 6,

SEQ ID NO: 6 R1-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-R2-Thr-R3-R4-Thr-Xaa₀₋₃₅-Gly-Ile-Val-R5-Gln-Cys-Cys-R6-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys- Asn;wherein R1 is His or Phe; wherein R2 is Tyr or Phe, R3 is Pro, Lys, orAsp; wherein R4 is Lys or Pro; wherein R5 is His or Glu; wherein R6 isHis or Thr; and wherein Xaa₀₋₃₅ is 0-35 of any amino acid or a break inthe amino acid chain;

and further wherein at least one substitution selected from the group ofthe following amino acid substitutions is present:

R1 is His; and

R6 is His; and

R5 and R6 together are His.

The 4Cl-Phe^(B24) substitution may also be introduced into a singlechain insulin analogue as disclosed in co-pending U.S. patentapplication Ser. No. 12/419,169, the disclosure of which is incorporatedby reference herein.

It is also envisioned that the chlorinated-Phe^(B24) substitution may beintroduced into an insulin analogue containing substitutions in theinsulin A-chain. For example, an insulin analogue may additionallycontain a lysine, histidine or arginine substitution at position A8, asshown in SEQ ID NO: 7, instead of the wild type threonine at position A8(see SEQ ID NO: 2).

SEQ ID NO: 7 Gly-Ile-Val-Glu-Gln-Cys-Cys-Xaa-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn [Xaa = His, Arg, or Lys]

As mentioned above, the 4Cl-Phe^(B24) substitution may be introducedwithin an engineered insulin monomer of high activity, designatedDKP-insulin, which contains the substitutions Asp^(B10) (D), Lys^(B28)(K) and Pro^(B29) (P). These three substitutions on the surface of theB-chain are believed to impede formation of dimers and hexamers. Use ofan engineered monomer circumvents confounding effects of self-assemblyon stability assays. The structure of DKP-insulin closely resembles acrystallographic protomer. The sequence of the B-chain polypeptide forDKP insulin is provided as SEQ ID NO: 8.

(DKP B-Chain Sequence) SEQ ID NO: 8Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-Asp-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- Phe-Tyr-Thr-Lys-Pro-Thr

Analogues of KP-insulin may be prepared by trypsin-catalyzedsemi-synthesis and purified by high-performance liquid chromatography(Mirmira, R. G., and Tager, H. S., 1989. J. Biol. Chem. 264: 6349-6354.)This protocol employs (i) a synthetic octapeptide representing residues(N)-GF*FYTKPT (including modified residue (F*) and “KP” substitutions(underlined); SEQ ID NO: 9) and (ii) truncated analoguedes-octapeptide[B23-B30]-insulin (DOI; SEQ ID NO: 15). Because theoctapeptide differs from the wild-type B23-B30 sequence (GFFYTPKT; SEQID NO: 10) by interchange of Pro^(B28) and Lys^(B29) (italics),protection of the lysine ε-amino group is not required during trypsintreatment. In brief under this protocol, des-octapeptide insulin (150mg) and octapeptide (150 mg) is dissolved in a mixture ofdimethylacetamide/1,4-butandiol/0.2 M Tris acetate (pH 8) containing 10mM calcium acetate and 1 mM ethylene diamine tetra-acetic acid (EDTA)(35:35:30, v/v, 4 mL). The 5-fold molar excess of octapeptide over DOIensures that the reverse reaction of trypsin (proteolytic direction) isprevented by substrate saturation. The final pH is adjusted to 7.0 with0.1 mL of N-methylmorpholine. The solution is cooled to 12° C., and 1.5mg of trypsin is added and incubated for 2 days at 12° C. An additional1.5 mg of trypsin is added after 24 hr. The reaction is acidified with0.1% trifluoroacetic acid and purified by preparative reverse-phase HPLC(C4). The product may then be verified by mass spectrometry usingmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF;Applied Biosystems, Foster City, Calif.). The general protocol forsolid-phase synthesis is as described (Merrifield et al., 1982.Biochemistry 21: 5020-5031). 9-fluoren-9-yl-methoxy-carbonyl(F-moc)-protected phenylalanine analogues are available from Chem-ImpexInternational (Wood Dale, Ill.).

Circular dichroism (CD) spectra may be obtained at 4° C. and 25° C.using an Aviv spectropolarimeter (Weiss et al., Biochemistry 39:15429-15440). Samples may contain ca. 25 μM KP-insulin or analogues in50 mM potassium phosphate (pH 7.4); samples are diluted to 5 μM forguanidine-induced denaturation studies at 25° C. To extract freeenergies of unfolding, denaturation transitions are fitted by non-linearleast squares to a two-state model as described by Sosnick et al.,Methods Enzymol. 317: 393-409. In brief, CD data θ(x), where x indicatesthe concentration of denaturant, are fitted by a nonlinear least-squaresprogram according to

${\theta(x)} = \frac{\theta_{A} + {\theta_{B}{\mathbb{e}}^{{({{{- \Delta}\; G_{H_{2}O}^{o}} - {mx}})}/{RT}}}}{1 + {\mathbb{e}}^{{- {({{\Delta\; G_{H_{2}O}^{o}} - {mx}})}}/{RT}}}$

where x is the concentration of guanidine and where θ_(A) and θ_(B) arebaseline values in the native and unfolded states. Baselines areapproximated by pre- and post-transition lines θ_(A)(x)=θ_(A) ^(H) ²^(O)+m_(A)x and θ_(B)(x)=θ_(B) ^(H) ² ^(O)+m_(B)x.

Relative activity is defined as the ratio of analogue to wild-type humaninsulin required to displace 50 percent of specifically bound ¹²⁵I-humaninsulin. A human placental membrane preparation containing the insulinreceptor (IR) is employed, as known in the art. Membrane fragments(0.025 mg protein/tube) were incubated with ¹²⁵I-labeled insulin (ca.30,000 cpm) in the presence of selected concentrations of unlabelledanalogue for 18 hours at 4° C. in a final volume of 0.25 ml of 0.05 MTris-HCl and 0.25 percent (w/v) bovine serum albumin at pH 8. Subsequentto incubation, mixtures are diluted with 1 ml of ice-cold buffer andcentrifuged (10,000 g) for 5 min at 4° C. The supernatant is thenremoved by aspiration, and the membrane pellet counted forradioactivity. Data is corrected for nonspecific binding (amount ofradioactivity remaining membrane associated in the presence of 1 μMhuman insulin. In all assays the percentage of tracer bound in theabsence of competing ligand was less than 15% to avoid ligand-depletionartifacts. An additional insulin receptor-binding assay to monitorchanges in activity during the course of incubation of the insulinanalogue at 37° C. may be performed using a microtiter plate antibodycapture as known in the art. Microtiter strip plates (Nunc Maxisorb) areincubated overnight at 4° C. with AU5 IgG (100 μl/well of 40 μg/ml inphosphate-buffered saline). Binding data may be analyzed by a two-sitesequential model. A corresponding microtiter plate antibody assay usingthe IGF Type I receptor may be employed to assess cross-binding to thishomologous receptor.

Modified residues were introduced within the context of KP-insulin.Activity values shown are based on ratio of hormone-receptordissociation constants relative to human insulin; the activity of humaninsulin is thus 1.0 by definition. Standard errors in the activityvalues were in general less than 25%. Free energies of unfolding(ΔG_(u)) at 25° C. were estimated based on a two-state model asextrapolated to zero denaturant concentration. Lag time indicates time(in days) required for initiation of protein fibrillation on gentleagitation at 37° C. in zinc-free phosphate-buffered saline (pH 7.4).

The chlorinated-Phe^(B24) substitution provided herein may be made ininsulin analogues such as lispro insulin (that is, an insulin analoguealso containing the substitutions Lys^(B28), Pro^(B29) (sold under thename Humalog®)). Such an insulin analogue is designatedchlorinated-Phe^(B24)-KP-insulin. For comparative purposes, fluorinesubstitutions were also introduced, essentially as described above, withthe exception of para-fluorinated phenylalanine being introduced atpositions B24 and B26 in lispro insulin. Analogues containingpara-fluorinated phenylalanine substitutions at position B24 aredesignated 4F-Phe^(B24)-KP-insulin. Analogues containingpara-fluorinated phenylalanine substitutions at position B26(substituting for tyrosine) are designated 4F-Phe^(B26)-KP-insulin.Designations of respective analogues of KP-insulin and DKP-insulin maybe abbreviated in tables and figures as KP and DKP with “insulin”omitted for brevity.

For chlorinated phenylalanine substitutions at B24, a syntheticoctapeptide representing residues (N)-GF*FYTKPT (chlorinatedphenylalanine residue indicated as “F*” and “KP” substitutions(underlined); SEQ ID NO: 9) and truncated analoguedes-octapeptide[B23-B30]-insulin (wild type at position B10, SEQ ID NO:15) were used. For fluorinated phenylalanine substitutions at B26, asynthetic octapeptide representing residues (N)-GFFF*TKPT (fluorinatedphenylalanine indicated again as “F*” and “KP” substitutions(underlined); SEQ ID NO: 16) and truncated analoguedes-octapeptide[B23-B30]-insulin (SEQ ID NO: 15) were used.

The resulting data for substitution of a halogenated phenylalanine atposition B24 or B26 in a lispro insulin analogue background arepresented below in Table 1.

TABLE 1 Stability And Activity Of Halogenated-Phe Analogues Of LisproInsulin ΔG_(u) ΔΔG_(u) C_(mid) m receptor Sample (Kcal/mol) (Kcal/mol)(M Gu-HCl) (Kcal/mol/M) binding (%) KP-insulin  3.0 ± 0.06 / 4.5 ± 0.10.61 ± 0.01 92 4F-Phe^(B24)-KP 2.75 ± 0.1  0.05 ± 0.16 4.4 ± 0.1 0.62 ±0.02 32 4F-Phe^(B26)-KP 3.7 ± 0.1  0.9 ± 0.16 4.9 ± 0.2 0.75 ± 0.02 134Cl-Phe^(B24)-KP 2.6 +/− 0.1 0.4 ± 0.2 4.7 +/− 0.2 0.58 +/− 0.02 90-100

Fibrillation Assays. The physical stability of 4Cl-Phe^(B24)-KP-insulinwas evaluated in triplicate during incubation in zinc-freephosphate-buffered saline (PBS) at pH 7.4 at 37° C. under gentleagitation in glass vials. The samples were observed for 12 days at aprotein concentration of 60 μM for visual appearance of cloudiness;twice daily aliquots were withdrawn for analysis of thioflavin-T (ThT)fluorescence. Because ThT fluorescence is negligible in the absence ofamyloid but is markedly enhanced on onset of fibrillation, this assayprobes for the lag time. Respective lag times for human insulin,KP-insulin, and 4Cl-Phe^(B24)-KP-insulin are 5±1 days, 3±1 days, andmore than 12 days. 4Cl-Phe^(B24)-KP-insulin is therefore at least 4-foldmore resistant to fibrillation under these conditions than is KP-insulinand at least 2-fold more resistant than human insulin. While not wishingto condition patentability on theory, it is envisioned that increasedfibrillation resistance of 4Cl-Phe^(B24) insulin analogues will allowthem to be formulated in a zinc-free formulation to enhance thefast-acting nature of the insulin analogue without significantlyshortening the storage time of a sample of the analogue, either beforeor after an individual sample has begun to be used.

Thermodynamic Stability. We measured the free energy of unfolding of4Cl-Phe^(B24)-KP-insulin relative to KP-insulin and LysA8-KP-insulin ina zinc-free buffer at pH 7.4 and 25° C. (10 mM potassium phosphate and50 mM KCl). This assay utilized CD detection of guanidine-induceddenaturation as probed at helix-sensitive wavelength 222 nm. Values ofΔGu were extrapolated to zero denaturant concentration to obtainestimates by the free energy of unfolding on the basis of a two-statemodel. Whereas the substitution ThrA8→Lys augmented thermodynamicstability by 0.6±0.2 kcal/mole, the 4Cl-Phe^(B24) modification decreasedstability by 0.4±0.2 kcal/mole.

The affinity of 4Cl-Phe^(B24)-KP-insulin-A8T for thedetergent-solubilized and lectin purified insulin receptor (isoform B)is similar to that of human insulin. A competitive displacement assayusing ¹²⁵I-labeled human insulin as tracer is shown in FIG. 3A usingisolated insulin receptor (isoform B): human insulin (triangles),KP-insulin (squares), 4Cl-Phe^(B24)-KP-insulin (inverted triangles). Allthree curves are closely aligned, indicating similar receptor-bindingaffinities. The affinity of 4Cl-Phe^(B24)-KP-insulin for the insulinreceptor was indistinguishable from that of KP-human insulin, in eachcase slightly lower than the affinity of wild-type insulin.

FIG. 3B shows results of corresponding assays employing Insulin-likeGrowth Factor I Receptor (IGF-1R), probed by competitive displacementusing ¹²⁵I-labeled IGF-I as tracer. Symbols are the same with theaddition of native IGF-I (circles). The rightward shift of the4Cl-Phe^(B24)-KP-insulin curve indicates decreased cross-binding toIGF-1R. The cross-binding of 4Cl-Phe^(B24)-KP-insulin to IGF-1R isreduced by approximately 3-fold relative to that of KP-insulin orwild-type insulin.

A similar comparison between human insulin (solid black line),KP-insulin (dashed line), 4Cl-Phe^(B24)-KP-insulin (triangles),4F-Phe^(B24)-KP-insulin (squares) using isolated insulin receptor(isoform B), is shown in FIG. 3C. The rightward shift of the4F-Phe^(B24)-KP-insulin curve relative to 4Cl-Phe^(B24)-KP-insulin, wildtype human insulin and lispro insulin shows decreased receptor-bindingaffinity with the use of a different halogen at the same position on thephenylalanine ring in comparison to a para-chloro substitution of thephenylalanine at B24. In contrast, the insulin receptor-binding affinityof 4Cl-Phe^(B24)-KP-insulin is similar to that of wild type humaninsulin and lispro (KP) insulin.

A further comparison between human insulin (solid line), KP-insulin(dashed line), 4Cl-Phe^(B26)-KP-insulin (triangles)4F-Phe^(B26)-KP-insulin (squares) using isolated insulin receptor(isoform B), is shown in FIG. 3D. Both 4Cl-Phe^(B26)-KP-insulin and4F-Phe^(B26)-KP-insulin show decreased insulin receptor-binding affinityin comparison to wild type and lispro insulins. As stated above,4Cl-Phe^(B24)-KP-insulin does not show a similar decrease inreceptor-binding affinity.

The in vivo potency of 4Cl-Phe^(B24)-KP-insulin in diabetic rats issimilar to that of KP-insulin. To enable characterization of biologicalactivity, male Lewis rats (˜300 g body weight) were rendered diabeticwith streptozotocin. Human insulin, KP-insulin, and4Cl-Phe^(B24)-KP-insulin were purified by HPLC, dried to powder, anddissolved in insulin diluent (Eli Lilly Corp). Rats were injectedsubcutaneously at time=0 with either 20 μg or 6.7 μg of KP-insulin or4Cl-Phe^(B24)-KP-insulin in 100 μl of diluent; the higher dose is at theplateau of the wild-type insulin dose-response curve whereas the lowerdose corresponds to 50-70% maximal initial rate of glucose disposal.Injection of diluent alone was performed as a negative control. 8 ratswere studies in each group. Blood was obtained from clipped tip of thetail at time 0 and at successive intervals up to 120 min. Blood glucosewas measured using a Hypoguard Advance Micro-Draw meter. Blood glucoseconcentrations were observed to decrease as shown in FIG. 4. The initialrate of fall of the blood glucose concentration during the first 24 minafter injection are similar on comparison of 4Cl-Phe^(B24)-KP-insulin(−225±29 mg/dl/h), KP-insulin (−256±35 mg/dl/h), and human insulin(−255±35 mg/dl/h). Any differences in initial rate are not statisticallysignificant. The duration of action of 4Cl-Phe^(B24)-KP-insulin over thenext 60 min appears shorter, however, than the durations of humaninsulin or KP-insulin.

Given the native receptor-binding affinity of 4Cl-Phe^(B24)-KP-insulin,it would be unusual for its potency to be less than that of humaninsulin. Indeed, insulin analogs with relative affinities in the range30-100% relative to wild-type typically exhibit native potencies invivo. It is formally possible, however, that the biological potency of4Cl-Phe^(B24)-KP-insulin is somewhat lower (on a molar basis) than thepotencies of human insulin or KP-insulin. If so, we note that any suchdecrease would be within the threefold range of the molar activities ofcurrent insulin products in clinical use (by convention respectiveinternational units (IU) are redefined to reflect extent of glucoselowering, leading to product-to-product differences in the number ofmilligrams or nanomoles per unit). It should be noted that the slowdecline in blood glucose concentration on control injection ofprotein-free diluent (brown dashed line in FIG. 4) reflects diurnalfasting of the animals following injection.

A surrogate marker for the pharmacokinetics of insulin hexamerdisassembly (designated the EDTA sequestration assay) employs cobaltions (Co²⁺) rather than zinc ions (Zn²⁺) to mediate hexamer assembly.Although Co²⁺ and Zn²⁺ hexamers are similar in structure, the cobalt ionprovides a convenient spectroscopic probe due to its unfilledd-electronic shell.

The principle of the assay is as follows. Solutions of R₆phenol-stabilized Co²⁺ insulin hexamers are blue due to tetrahedral Co²⁺coordination; on disassembly the protein solution is colorless asoctahedral Co²⁺ coordination by water or EDTA(ethylene-diamine-tetra-acetic acid; a strong chelator of metal ions)lacks optical transitions at visible wavelengths as a consequence ofligand field theory. The EDTA sequestration assay exploits thesespectroscopic features as follows. At time t=0 a molar excess of EDTA isadded to a solution of R₆ insulin hexamers or insulin analog hexamers.Although EDTA does not itself attack the hexamer to strip it of metalions, any Co²⁺ ions released in the course of transient hexamerdisassembly become trapped by the chelator and thus unavailable forreassembly. The rate of disappearance of the blue color (the tetrahedrald-d optical transition at 574 nm of the R-specific insulin-bound Co²⁺)thus provides an optical signature of the kinetics of hexamerdisassembly.

Averaged traces of insulin cobalt solutions showing characteristicspectral profiles from 400-750 nm were determined before and afteraddition of 2 mM EDTA (FIGS. 5A-C). Samples were dissolved in 50 mM Tris(pH 7.4), 50 mM phenol, and 0.2 mM CoCl₂. NaSCN was then added to afinal concentration of 1 mM. The kinetics of hexamer dissociation afteraddition of 2 mM EDTA as monitored at 574 nm (25° C. and pH 7.4) arealso shown. The spectra of the analogues before EDTA extraction areshown as solid lines. Post-EDTA extraction, the spectra are displayed asdashed lines. Wild type is shown in Panel A, KP-insulin in Panel B, and4Cl-Phe^(B24)-KP-insulin as in Panel C. Data were normalized to timezero for each sample.

On the one hand, the baseline optical absorption spectra of thehexameric cobalt complexes at t=0 are similar among wild-type insulinhexamers, KP insulin hexamers, and 4Cl-Phe^(B24)-KP-insulin hexamers(see FIGS. 5A-5C). The similar shapes and magnitudes of these respectived-d electronic transitions imply that the metal ions are in similarR₆-specific tetrahedral coordination sites in wild-type and varianthexamers. This result is significant as it implies that4Cl-Phe^(B24)-KP-insulin remains competent for metal-ion-mediatedassembly and hence a zinc-based formulation.

The kinetics of hexamer dissociation after addition of 2 mM EDTA asmonitored at 574 nm (25° C. and pH 7.4) shows that the wild-type andvariant hexamers exhibit marked differences in rates of EDTA-mediatedCo²⁺ sequestration. As expected, the wild-type hexamer exhibits thegreatest kinetic stability (solid line in FIG. 5D), followed byKP-insulin (dashed-dotted line in FIG. 5D), and 4-Cl-PheB24-KP-insuln(dotted line in FIG. 5D). Respective half-lives are 481 sec (wild type),363 sec (KP-insulin), and 66 sec (4Cl-Phe^(B24)-KP-insulin). The extentof acceleration induced by the para-chloro-aromatic substitution is thusmore profound than that associated with the “KP switch” of Lisproinsulin (Humalog™). Because diffusion of zinc ions from the site ofsubcutaneous injection is analogous to the in vitro sequestration ofcolbalt ions in the EDTA Sequestration assay, these findings predictthat 4Cl-Phe^(B24)-KP-insulin will exhibit a marked acceleration ofabsorption.

The pharmacokinetic (PK) and pharacodynamic (PD) properties and potencyof 4-Cl-Phe^(B24)-KP-insulin were investigated in relation to wild-typeinsulin (Humulin™; Eli Lilly and Co.) and KP-insulin (Humalog™) inadolescent Yorkshire farm pigs (weight 25-45 kg). The wild type andKP-insulin were used as provided by the vendor (Eli Lilly and Co.) inU-100 strength. The 4-Cl-Phe^(B24)-KP-insulin was formulated in Lillydiluent with a ratio of protein to zinc ions similar to that of the wildtype and KP-insulin products; its strength was U-87. On the day ofstudy, each animal underwent anesthesia induction with Telazol and thengeneral anesthesia with isoflurane. Each animal was endotreacheallyintubated, and oxygen saturation and end-tidal expired CO₂ werecontinuously monitored. To block endogenous pancreatic α- and β-cellsecretion, pigs were given a subcutaneous injection of octreotideacetate (44 μg/kg) approximately 30 min before beginning the clamp studyand every 2 h thereafter. After IV catheters were placed and baselineeuglycemia was established with 10% dextrose infusion, an IV injectionof the insulin was given through the catheter. In order to quantifyperipheral insulin-mediated glucose uptake, a variable-rate glucoseinfusion was given to maintain a blood glucose concentration ofapproximately 85 mg/dl. Such a glucose infusion was typically requiredfor 5-8 h until the glucose infusion rate returned to the pre-insulinbaseline. Glucose concentrations were measured with a Hemocue 201portable glucose analyzer every 10 min (instrument error rate: 1.9%).The computerized protocol for glucose clamping was as described byMatthews et al. 2-ml blood samples for insulin assay was also obtainedaccording to the following schedule: from 0-40 min after insulindelivery: 5-minute intervals; from 50-140 min: 10-minute intervals, andfrom 160 min—to the point when GIR is back to baseline: 20-minintervals. For analysis of PK/PD, a 20-min moving mean curve fit andfilter was applied. PD was measured as time to early half-maximal effect(½ T_(max) Early), time to late half-maximal effect (½ T_(max) Late),time to maximal effect, and area-under-the-curve (AUC) over baseline.For each of these analyses, the fitted curve, not the raw data, wasused. Each of 3 pigs underwent 3 studies. The results of these studiesare provided in FIGS. 6-9.

4-Cl-Phe^(B24)-KP-insulin (abbreviated in FIG. 6 as 4-Cl-Lispro Insulin)was found to exhibit a significantly less prolonged late “tail” thanKP-insulin or wild-type insulin. The improved turn-off of insulin actionsuggests a potential clinical benefit with regard to late post-prandialhypoglycemia.

FIG. 7 summarizes 20 pharmacodynamic studies in pigs demonstratingsignificant improvement in ½ T-max late in 4Cl-Phe^(B24)-KP-insulin overKP-insulin at five different dosing levels, 0.05 U/kg, 0.1 U/kg, 0.2U/kg, 0.5 U/kg, and 1 U/kg.

FIG. 8 summarizes 14 pharmacodynamic studies in pigs suggestingimprovement in ½ T-max early in 4Cl-Phe^(B24)-KP-insulin over KP-insulinat three different dosing levels, 0.05 U/kg, 0.1 U/kg, 0.2 U/kg.

FIG. 9 summarizes ten, matched pharmacodynamics studies comparing therelative potencies 4Cl-Phe^(B24)-KP-insulin with that of KP insulin asmeasured by area under the curve (AUC) in which the slightly reducedaverage potency for 4-Cl-KP was found not to be statisticallysignificant (p=0.22). The pharmacokinetic (PK) and pharmacodynamic (PD)properties of 4-Cl-Phe^(B24)-KP-insulin in relation to wild-type insulinand KP-insulin (Lispro-insulin) under similar formulation conditions(zinc insulin hexamers or zinc insulin analog hexamers stabilized byphenol and meta-cresol) show that the potency of4-Cl-Phe^(B24)-KP-insulin, as measured by area-under-the-curve (AUC)method, was similar to those of wild-type insulin and KP-insulin.

A method for treating a patient comprises administering achlorinated-Phe^(B24) substituted insulin analogue to the patient. Inone example, the insulin analogue is a 4Cl-Phe^(B24)-KP insulin. Theinsulin analogue may optionally contain a histidine, lysine or argininesubstitution at position A8. In another example, thechlorine-substituted insulin analogue additionally contains one or moresubstitutions elsewhere in the insulin molecule designed to alter therate of action of the analogue in the body. In still another example,the insulin analogue is administered by an external or implantableinsulin pump. An insulin analogue of the present invention may alsocontain other modifications, such as a tether between the C-terminus ofthe B-chain and the N-terminus of the A-chain as described more fully inco-pending U.S. patent application Ser. No. 12/419,169.

A pharamaceutical composition may comprise such insulin analogues andmay optionally include zinc. Zinc ions may be included in such acomposition at a level of a molar ratio of between 2.2 and 3.0 perhexamer of the insulin analogue. In such a formulation, theconcentration of the insulin analogue would typically be between about0.1 and about 3 mM; concentrations up to 3 mM may be used in thereservoir of an insulin pump. Modifications of meal-time insulinanalogues may be formulated as described for (a) “regular” formulationsof Humulin® (Eli Lilly and Co.), Humalog® (Eli Lilly and Co.), Novalin®(Novo-Nordisk), and Novalog® (Novo-Nordisk) and other rapid-actinginsulin formulations currently approved for human use, (b) “NPH”formulations of the above and other insulin analogues, and (c) mixturesof such formulations. As mentioned above, it is believed that theincreased resistance to fibrillation will permit4Cl-Phe^(B24)-containing insulin analogues to be formulated without thepresence of zinc to maximize the fast acting nature of the analogue.However, it is also believed that even in the presence of zinc, the4Cl-Phe^(B24)-containing insulin analogues will dissociate from hexamersinto dimers and monomers sufficiently quickly as to also be considered afast-acting insulin analogue formulation.

Excipients may include glycerol, glycine, other buffers and salts, andanti-microbial preservatives such as phenol and meta-cresol; the latterpreservatives are known to enhance the stability of the insulin hexamer.Such a pharmaceutical composition may be used to treat a patient havingdiabetes mellitus or other medical condition by administering aphysiologically effective amount of the composition to the patient.

A nucleic acid comprising a sequence that encodes a polypeptide encodingan insulin analogue containing a sequence encoding at least a B-chain ofinsulin with a chlorinated phenylalanine at position B24 is alsoenvisioned. This can be accomplished through the introduction of a stopcodon (such as the amber codon, TAG) at position B24 in conjunction witha suppressor tRNA (an amber suppressor when an amber codon is used) anda corresponding tRNA synthetase, which incorporates a non-standard aminoacid into a polypeptide in response to the stop codon, as previouslydescribed (Furter, 1998, Protein Sci. 7:419-426; Xie et al., 2005,Methods. 36: 227-238). The particular sequence may depend on thepreferred codon usage of a species in which the nucleic acid sequencewill be introduced. The nucleic acid may also encode other modificationsof wild-type insulin. The nucleic acid sequence may encode a modified A-or B-chain sequence containing an unrelated substitution or extensionelsewhere in the polypeptide or modified proinsulin analogues. Thenucleic acid may also be a portion of an expression vector, and thatvector may be inserted into a host cell such as a prokaryotic host celllike an E. coli cell line, or a eukaryotic cell line such as S.cereviciae or Pischia pastoris strain or cell line.

For example, it is envisioned that synthetic genes may be synthesized todirect the expression of a B-chain polypeptide in yeast Piscia pastorisand other microorganisms. The nucleotide sequence of a B-chainpolypeptide utilizing a stop codon at position B24 for the purpose ofincorporating a chlorinated phenylalanine at that position may be eitherof the following or variants thereof:

(a) with Human Codon Preferences: (SEQ ID NO: 11)TTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTAGTTCTACACACCCAAGACC(b) with Pichia Codon Preferences: (SEQ ID NO: 12)TTTGTTAACCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTAGTTTTACACTCCAAAGACT

Similarly, a full length pro-insulin cDNA having human codon preferencesand utilizing a stop codon at position B24 for the purpose ofincorporating a chlorinated phenylalanine at that position may have thesequence of SEQ ID NO: 13.

(SEQ ID NO: 13) TTTGTGAACC AACACCTGTG CGGCTCACAC CTGGTGGAAGCTCTCTACCT AGTGTGCGGG GAACGAGGCT AGTTCTACACACCCAAGACC CGCCGGGAGG CAGAGGACCT GCAGGTGGGGCAGGTGGAGC TGGGCGGCGG CCCTGGTGCA GGCAGCCTGCAGCCCTTGGC CCTGGAGGGG TCCCTGCAGA AGCGTGGCATTGTGGAACAA TGCTGTACCA GCATCTGCTC CCTCTACCAG CTGGAGAACT ACTGCAACTA G

Likewise, a full length human pro-insulin cDNA utilizing a stop codon atposition B24 for the purpose of incorporating a chlorinatedphenylalanine at that position and having codons preferred by P.pastoris may have the sequence of SEQ ID NO: 14

(SEQ ID NO: 14) TTTGTTAACC AACATTTGTG TGGTTCTCAT TTGGTTGAAGCTTTGTACTT GGTTTGTGGT GAAAGAGGTT AGTTTTACACTCCAAAGACT AGAAGAGAAG CTGAAGATTT GCAAGTTGGTCAAGTTGAAT TGGGTGGTGG TCCAGGTGCT GGTTCTTTGCAACCATTGGC TTTGGAAGGT TCTTTGCAAA AGAGAGGTATTGTTGAACAA TGTTGTACTT CTATTTGTTC TTTGTACCAA TTGGAAAACT ACTGTAACTA A

Based upon the foregoing disclosure, it should now be apparent thatchlorine-substituted insulin analogues will carry out the objects setforth hereinabove. Namely, these insulin analogues exhibit enhancedthermodynamic stability, resistance to fibrillation and potency inreducing blood glucose levels. The chlorine substitutedphenylalanine-containing insulin analogues also have reducedcross-reactivity to insulin-like growth factor (IGFR). It is, therefore,to be understood that any variations evident fall within the scope ofthe claimed invention and thus, the selection of specific componentelements can be determined without departing from the spirit of theinvention herein disclosed and described.

The following literature is cited to demonstrate that the testing andassay methods described herein would be understood by one of ordinaryskill in the art.

Furter, R., 1998. Expansion of the genetic code: Site-directedp-fluoro-phenylalanine incorporation in Escherichia coli. Protein Sci.7:419-426.

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What is claimed is:
 1. A method of incorporating a chlorinatedphenylalanine into an insulin B-chain polypeptide, the methodcomprising: expressing a nucleic acid encoding the insulin B-chainpolypeptide, wherein the chlorinated phenylalanine is encoded by a stopcodon, wherein the nucleic acid is expressed in conjunction with asuppressor tRNA and a corresponding tRNA synthetase which incorporatesthe chlorinated phenylalanine in response to the stop codon, wherein thechlorinated phenylalanine is incorporated into the insulin B-chainpolypeptide at position B24 relative to wild type insulin, and whereinthe chlorinated phenylalanine is para-monochloro-phenylalanine.
 2. Themethod of claim 1 wherein the stop codon is TAG.
 3. The method of claim2, wherein the nucleic acid encoding the insulin B-chain polypeptidecomprises SEQ ID NO: 11 or SEQ ID NO:
 12. 4. The method of claim 2,wherein the nucleic acid encoding the insulin B-chain polypeptidecomprises nucleotides 61-90 of SEQ ID NO: 11 or nucleotides 61-90 of SEQID NO:
 12. 5. The method of claim 1, wherein the nucleic acid is aportion of an expression vector.
 6. The method of claim 5, wherein theexpression vector is inserted into a host cell.
 7. A method of producingan insulin analogue containing a chlorinated phenylalanine, the methodcomprising expressing a nucleic acid encoding at least amino acidresidues 20-30 of the insulin B-chain polypeptide, wherein thechlorinated phenylalanine encoded by a stop codon and wherein thenucleic acid is expressed in conjunction with a suppressor tRNA and acorresponding tRNA synthetase which incorporates the chlorinatedphenylalanine in response to the stop codon, and wherein the chlorinatedphenylalanine is para-monochloro-phenylalanine located at position B24relative to wild type insulin.